Lecture 2 - How To Examine Cells And Tissues Flashcards
What is the definition of a Tissue?
(Short definition)
Tissue is a Latin word that means woven
What is the definition of a Tissue?
(Long definition)
It is a collection of cells that are adapted to perform a specific function
What are the 4 types of tissue?
-Epithelial
-Connective
-Muscle
-Nervous
What is the definition of epithelial tissue?
Sheets of contiguous cells, of varied embryonic origins, that cover the external surface of the body and line inertial surfaces including the bodies vessels
Give an example of epithelial tissue
The outer layer of the skin
Lining of the small intestine
What does connective tissue consist of?
Cells and extracellular proteins/glycoproteins and gels
What are the main cells found in connective tissues?
Fibroblasts
Chondrocytes
Osteoblasts/osteocytes/osteoclasts
Stem cells/progenitor cells
What are the main products of connective tissues?
Fibres
Ground substance
What does ground substance consist of?
Cells
Extracellular matric
What are the 3 types of muscle tissue?
Skeletal
Cardiac
Smooth
What is the function of nerve tissue?
Fast communication system of the body
What is the relationship between millimetres and micrometers?
There are a 1000 micrometers in 1mm
Or a millimetre is x1000 larger than a micrometer
What is the relationship between micrometers and nanometers?
There are 1000 nanometers in a micrometer
Or a micrometer is x1000 times larger than a nanometer
What is the relationship between millimetres and nanometers?
There are 1000000 nanometers in 1mm
OR a millimetre is x1000000 larger than a nanometer
What is the definition for the Limit of Resolution?
The smallest distance by which 2 objects can be separated and still be clearly distinguishable as 2 separate objects
Which has better resolving power, light microscopes or electron microscopes?
Electron microscopes
What methods are there to obtain a tissue biopsy?
Surgery and then dissection
Scraping methods (curettes, scalpel scrapes)
Sharp needles (needle biopsy, pipelle)
Venupuncture (for blood smears)
What methods of tissue biopsy can be used to obtain an endometrial sample?
Scraping (endometrial curettage)
Sharp needle (Pipelle)
Surgery (hysterectomy)
What method can be used to obtain a bone marrow biopsy?
Sharp needle
Why does a specimen/biopsy require fixing/fixation?
To prevent digestion by microbes or being destroyed by decay
What does fixation remove from a specimen?
Water
Stiffens biopsy
What is the most common fixative that is used in preparing biopsies?
Buffered formalin solution
How can fixation cause biopsy shrinkage and formation of artefacts?
If a specimen is left in fixative for too long it becomes to dehydrated shrinking
Once a biopsy has been obtained and fixated, what is the next step?
Embedding
What happens in embedding?
Dehydration of specimen in different concs of alcohol
Then its immersed in hot paraffin wax which is allowed to solidify
What is the normal medium of choice for embedding?
Paraffin wax
What happens after embedding has occured?
Sectioning
Thinly sliced by microtome
What is usually done after a specimen has been sectioned/thinly sliced?
Staining
Give some examples of stains commonly used:
Haemotoxyin and Eosin (H&E)
Massons trichrome
Periodic Acid-Shiff stain
What does the Haematoxylin stain part of H&E stain and what to colour does it stain?
Stains nucleus BLUE most strongly
What does the Eosin component of H&E stain and what colour does it stain?
Stains alkali stuff (the proteins)
Cytoplasm and extracellular matrix stained PINK the most strongly
Why is H&E (haematoxylin and Eosin) useful?
Nuclei are clearer and more detailed cytoplasm
What is Periodic Acid-Shiff stain commonly used to stain and why?
Kidney
Stains anything with sugar attached pink
Lots of sugar in kidney
What is immunohistochemistry/immunostaining?
Using labelled antibodies which bind to specific tissues/cells so they can be easily examined
How does immunohistochemistry allow us to see specific cells or tissues?
A primary antibody that is specific to the antigen found on the specific cell/tissue we want to view binds to the cell of interest
A secondary labelled antibody is bound to the primary antibody. This antibody has a coloured dye bound to it which shows the presence of the specific antigen/cell
How does immunofluroescence work?
Labelled primary antibody with fluroscent tag attached binds to specific antigen on cell of interest
Signal is emitted onto the fluorescent tag, it fluoresces and is visible
What is the advantage of Staining specimens?
It creates clear contrast so a specimen can be viewed in detail
What is a frozen section?
Surgical specimen on metal disc rapidly frozen
Cut with microtome in a freezer
Stained with H&E
What is the advantages of a frozen section over a Paraffin wax formalin fixed specimen?
Prepared rapidly (used during surgeries)
Can be done to fresh tissues
What are the disadvantages of a frozen section?
Poor stain retention
Do not last very long (months)
Poor clarity
What are the advantages of a Paraffin wax formalin fixed specimen over a frozen section?
Last forever
Clear under microscope
Used for diagnosis
How are tissues prepared for electron microscopy?
Fixed with Glutaraldehyde
Embed in Epoxy resin
Stain osmium tetroxide
How are tissues prepared for electron microscopy?
Fixed with Glutaraldehyde
Embed in Epoxy resin
Stain osmium tetroxide
What are the differences between light a electron microscopy?
L = normal colours E = monochrome
L = cheap easy prep E = Expensive difficult
L = living and moving specimens E = Dead and inert
L = lower mag and res E = Higher mag and res
What does resolution of a microscope depend on?
The wavelength of the radiation it uses
Why do electron microscopes have a better/smaller limit of resolution than light microscopes?
They use electrons which have a smaller wavelength than light
What are the 2 types of electron microscopy?
TEM (Transition Electron Microscopy)
SEM (Scanning Electron Microscopy)
What is SEM good for?
Viewing 3D images of the surfaces of specimens
What is TEM good for?
Viewing 2D images of Intracellular organelles
What is confocal microscopy?
A laser excites a fluorescent dye in specimen
Light emitted passes through pinhole to detector
What are the advantages of confocal microscopy?
Entire depth of tissue can be examined
3D images can be created
Can evaluate and lead to rapid diagnoses of eye diseases
What is the advantage of Phase contrast microscopy?
Living cells can be viewed in their natural stated since fixing and embedding is not required
What is dark field microscopy?
Illuminating a live sample with light that isn’t collected by the objects lens. This makes the black background with bright objects on it
What are the advantages of Dark field microscopy?
Creates an extremely clear contrast
Easy to do
What stain is best to detect sugars/polysaccharides ?
Periodic acid-schiff reagent
What is the simple order of preparing a tissue sample for H&E staining?
Fixation
Embedding
Cutting/microtomy
Staining
Mounting
What type of histiological technique is likely to be used to identify presence or absence of a tumour?
Haematoxylin and Eosin Staining (H&E)
